MicroRNAs (miRNAs) are evolutionary conserved small RNAs that post‐transcriptionally regulate the expression of target genes. To date, the role of miRNAs in liver development is not fully understood. By using an experimental model that allows the induced and controlled differentiation of mouse fetal hepatoblasts (MFHs) into mature hepatocytes, we identified miR‐148a as a hepatospecific miRNA highly expressed in adult liver. The main finding of this study revealed that miR‐148a was critical for hepatic differentiation through the direct targeting of DNA methyltransferase (DNMT) 1, a major enzyme responsible for epigenetic silencing, thereby allowing the promotion of the “adult liver” phenotype. It was also confirmed that the reduction of DNMT1 by RNA interference significantly promoted the expression of the major hepatic biomarkers. In addition to the essential role of miR‐148a in hepatocyte maturation, we identified its beneficial effect through the repression of hepatocellular carcinoma (HCC) cell malignancy. miR‐148a expression was frequently down‐regulated in biopsies of HCC patients as well as in mouse and human HCC cell lines. Overexpressing miR‐148a led to an enhancement of albumin production and a drastic inhibition of the invasive properties of HCC cells, whereas miR‐148a silencing had the opposite consequences. Finally, we showed that miR‐148a exerted its tumor‐suppressive effect by regulating the c‐Met oncogene, regardless of the DNMT1 expression level. Conclusion: miR‐148a is essential for the physiology of the liver because it promotes the hepatospecific phenotype and acts as a tumor suppressor. Most important, this report is the first to demonstrate a functional role for a specific miRNA in liver development through regulation of the DNMT1 enzyme. (Hepatology 2013;53:1153–1165)

S-adenosylmethionine (SAMe) is involved in numerous complex hepatic processes such as hepatocyte proliferation, death, inflammatory responses, and antioxidant defense. One of the most relevant actions of SAMe is the inhibition of hepatocyte proliferation during liver regeneration. In hepatocytes, SAMe regulates the levels of cytoplasmic HuR, an RNAbinding protein that increases the half-life of target messenger RNAs such as cyclin D1 and A2 via inhibition of hepatocyte growth factor (HGF)-mediated adenosine monophosphateactivated protein kinase (AMPK) phosphorylation. Because AMPK is activated by the tumor suppressor kinase LKB1, and AMPK activates endothelial nitric oxide (NO) synthase (eNOS), and NO synthesis is of great importance for hepatocyte proliferation, we hypothesized that in hepatocytes HGF may induce the phosphorylation of LKB1, AMPK, and eNOS through a process regulated by SAMe, and that this cascade might be crucial for hepatocyte growth. We demonstrate that the proliferative response of hepatocytes involves eNOS phosphorylation via HGF-mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe and NO. We also show that knockdown of LKB1, AMPK, or eNOS with specific interference RNA (iRNA) inhibits HGF-mediated hepatocyte proliferation. Finally, we found that the LKB1/AMPK/eNOS cascade is activated during liver regeneration after partial hepatectomy and that this process is impaired in mice treated with SAMe before hepatectomy, in knockout mice deficient in hepatic SAMe, and in eNOS knockout mice. Conclusion: We have identified an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe, and NO that functions as a critical determinant of hepatocyte proliferation during liver regeneration after partial hepatectomy. (HEPATOLOGY 2009;49:608-617.)

Glycine N-methyltransferase (GNMT) catabolizes S-adenosylmethionine (SAMe), the main methyl donor of the body. Patients with cirrhosis show attenuated GNMT expression, which is absent in HCC samples. GNMT−/− mice develop spontaneous steatosis that progresses to steatohepatitis, cirrhosis and HCC. The liver is highly enriched with innate immune cells and plays a key role in the body’s host defense and in the regulation of inflammation. Chronic inflammation is the major hallmark of NASH progression. The aim of our study was to uncover the molecular mechanisms leading to liver chronic inflammation in the absence of GNMT, focusing on the implication of NK/NKT cells. We found increased expression of Th1- over Th2-related cytokines, TRAIL-R2/DR5 and several ligands of NK cells in GNMT−/− livers. Interestingly, NK cells from GNMT−/− mice were spontaneously activated; expressed more TRAIL and had strong cytotoxic activity, suggesting their contribution to the pro-inflammatory environment in the liver. Accordingly, NK cells mediated hypersensitivity to ConA-mediated hepatitis in GNMT−/− mice. Moreover, GNMT−/− mice were hypersensitive to endotoxin-mediated liver injury. NK cell depletion and adoptive transfer of TRAIL−/− liver-NK cells protected the liver against LPS-liver damage. Conclusions Our data allow us to conclude that TRAIL-producing NK cells actively contribute to promote a pro-inflammatory environment at early stages of fatty liver disease suggesting that this cell compartment may contribute to the progression of NASH.

SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.

Hepatic S-adenosylmethionine (SAMe) is maintained constant by the action of methionine adenosyltransferase I/III (MATI/III), which converts methionine into SAMe and glycine Nmethyltransferase (GNMT), which eliminates excess SAMe to avoid aberrant methylation reactions. During liver regeneration after partial hepatectomy (PH) MATI/III activity is inhibited leading to a decrease in SAMe. This injury-related reduction in SAMe promotes hepatocyte proliferation because SAMe inhibits hepatocyte DNA synthesis. In MATI/III-deficient mice, hepatic SAMe is reduced, resulting in uncontrolled hepatocyte growth and impaired liver regeneration. These observations suggest that a reduction in SAMe is crucial for successful liver regeneration. In support of this hypothesis we report that liver regeneration is impaired in GNMT knockout (GNMT-KO) mice. Liver SAMe is 50-fold higher in GNMT-KO mice than in control animals and is maintained constant following PH. Mortality after PH was higher in GNMT-KO mice than in control animals. In GNMT-KO mice, nuclear factor B (NF B), signal transducer and activator of transcription-3 (STAT3), inducible nitric oxide synthase (iNOS), cyclin D1, cyclin A, and poly (ADP-ribose) polymerase were activated at baseline. PH in GNMT-KO mice was followed by the inactivation of STAT3 phosphorylation and iNOS expression. NF B, cyclin D1 and cyclin A were not further activated after PH. The LKB1/AMPactivated protein kinase/endothelial nitric oxide synthase cascade was inhibited, and cytoplasmic HuR translocation was blocked despite preserved induction of DNA synthesis in GNMT-KO after PH. Furthermore, a previously unexpected relationship between AMPK phosphorylation and NF B activation was uncovered. Conclusion: These results indicate that multiple signaling pathways are impaired during the liver regenerative response in GNMT-KO mice, suggesting that GNMT plays a critical role during liver regeneration, promoting hepatocyte viability and normal proliferation. (HEPATOLOGY 2009;50:443-452.) M ethionine is an essential amino acid metabolized mainly by the liver where it is first converted to S-adenosylmethionine (SAMe) through a reaction catalyzed by the enzyme methionine adenosyltransferase I/III (MATI/III), and then to S-adenosylhomocysteine by the action of the enzyme glycine N-methyltransferase (GNMT). 1 MATI and MATIII are expressed mainly in the adult liver. They are, respectively, tetramers and dimers of the same single subunit encoded by the gene MAT1A. 2 A third isoenzyme, MATII, contains a catalytic subunit encoded by a second gene, MAT2A, and is expressed in all tissues including adult liver. 2 MAT1A knockout (MAT1A-KO) mice have elevated plasma levels of methionine and reduced hepatic SAMe content, 3 indicating that MATII cannot compensate for the loss of MATI/III. Although there are many enzymes that utilize SAMe in the liver, quantitatively the most important is GNMT. 1 Mice lacking functional Abbreviations: AMPK, AMP-activated protein kinase; BrdU, bromodeoxyuridine; CC, compound

Primary liver tumors are mainly represented by hepatocellular hepatocarcinoma (HCC), one of the most aggressive and resistant forms of cancer. Numerous studies have reported the key role of microRNAs (miRNAs) in development, cell proliferation, apoptosis, and tumor biology. The alteration of cancer-related miRNA expression can be associated with tumorigenesis. In HCC, deregulated miRNAs frequently act as oncogenes or altered tumor suppressors. Distinct subtypes of hepatic cancer can also be related to an aberrant expression of particular miRNAs, arguing for the significance of using miRNAs as tumor biomarkers in order to refine the HCC grading assessment. In this article, we review the latest reports regarding miRNA profiling and the potential of small RNAs in HCC diagnosis. The relevance of cancer-related miRNA signatures for the prognosis and better understanding of liver cancer outcome is then considered.

The gastric glands synthesize glycoproteins whose oligosaccharides are linked to the peptide core mainly by the O-glycosidic bond, specifically removed by beta-elimination procedure. Our aim was to research the possibility of the existence of two subtypes of O-linked oligosaccharides with a different behavior to the removal procedure. The lectins from peanut (PNA) and Maackia amurensis (MAA-I) were histochemically used as markers of the O-linked oligosaccharides. Sections were also pretreated with beta-elimination and/or peptide N-Glycosidase F (PNGase-F) for the specific removal of O- and N-linked oligosaccharides, respectively. The lectin GNA, which mainly labels to N-linked oligosaccharides, was used to test the correct working of PNGase-F. To test the possibility that the beta-elimination treatment could remove the terminal sialic acid residues, the lectin LFA was used. The surface epithelium was negative to PNA, while it became strongly positive when beta-elimination was performed for 1 day. This staining was resistant to PNGase-F, suggesting that PNA was labeling to O-linked oligosaccharides. However, after beta-elimination for 5 days this staining is not observed. A similar pattern appeared with MAA-I. We propose the existence of two subtypes of O-linked oligosaccharides: labile and resistant. The labile O-linked oligosaccharides are removed with beta-elimination for 1 day, unmasking the PNA-positive oligosaccharides. These oligosaccharides are resistant O-linked oligosaccharides because staining is abolished with longer treatment of beta-elimination. The results with MAA-I also support this suggestion. In summary, the labile O-linked oligosaccharides are removed with short treatment, while the resistant O-linked oligosaccharides need a stronger procedure (for 5 days).

BackgroundGolgi‐associated PDZ and coiled‐coil motif‐containing protein (GOPC) is a Golgi protein that plays a role in vesicular transport and intracellular protein trafficking and degradation. Mice deficient in GOPC protein have globozoospermia and are infertile. The germ cell nuclear factor (GCNF) is a member of the nuclear receptor superfamily which is expressed in male germ cells, from spermatocytes and spermatids, both in the nucleus and the acrosomal region. It is not known if its expression could be altered in Gopc−/− mice with defective acrosomes.ObjectivesThe aim of the present work was to analyze the distribution of GCNF protein in spermatids of Gopc−/− knockout mice.Materials and methodsWe have analyzed the expression and distribution during spermatogenesis of GCNF and its deregulation in Gopc−/− mutant mice by RT‐qPCR, Western blot, immunohistochemistry and immunogold.ResultsGerm cell nuclear factor was localized in the nucleus of all the cell types in the seminiferous tubules. Despite being a nuclear protein, it was also located in the acrosome and in the manchette of elongating spermatids. We have found that in the absence of GOPC, the expression of GCNF was increased in the nucleus of spermatocytes, mainly in leptotene, and in the nucleus and the manchette during the spermatid elongation.Discussion and ConclusionGopc−/− mice have defective acrosome and manchette. The manchette is involved in the transport of proteins through the cytoplasm and the nucleus. Considering that the GCNF protein is normally transported to the acrosome and the nucleus, it can be thought that transport deficiencies in Gopc−/− mice are responsible for the increased expression of this protein.